Influenza A viruses encode a non-structural protein termed NS1 that functions as a virulence determinant, especially in highly pathogenic H5N1 viruses. NS1 is a multifunctional protein that acts as an antagonist of multiple interferon-mediated antiviral mechanisms. Recent work has shown that NS1 proteins from avian influenza viruses, including H5N1 viruses, contain four carboxyl terminal residues termed the PDZ-binding domain (PDZ-BD) or PDZ-binding motif (PBM) that act as a virulence determinant through unknown mechanisms. We have recently found that the NS1 PBM of an avian H6N6 virus allows NS1 to associate with Dlg1 and Scribble, two cellular proteins that contain PDZ domains and regulate cellular polarity and signaling. The interaction between NS1 and Scribble is a direct protein-protein interaction. Our initial results also suggest that the NS1 PBM functions to protect infected cells from apoptosis and may also disrupt tight junctions between epithelial cells. We have also recently determined the crystal structure of the full-length NS1 protein from an H5N1 influenza virus along with cryo-EM imaging of NS1 (H1N1) bound to dsRNA. These studies have provided valuable insight into how NS1 can sequester variable lengths of dsRNA and make specific interactions with cellular proteins that mediate NS1 function. In our research plan, we propose to test the hypotheses that the interaction between NS1 with an avian virus PBM and Scribble and Dlg1 contributes to viral replication and pathogenesis through protecting infected cells from apoptosis and also perturbing tight junction integrity. We will use both functional and structural approaches to test these and other specific hypotheses about the function of the NS1 PBM. We have used reverse genetics to introduce the NS1 protein of an avian H6N6 virus into the background of the Udorn H3N2 strain. Importantly, experiments with this virus can be performed under BSL2 containment conditions, greatly facilitating an in-depth analysis of the PBM from an avian virus NS1 protein. This model system will allow us to elucidate mechanisms involved in the function of the NS1 PBM in a two year funding period, paving the way for future focused studies with the NS1 from H5N1 viruses which will require higher containment levels. PUBLIC HEALTH RELEVANCE: The NS1 protein of influenza A viruses contributes to viral pathogenesis, especially in current highly pathogenic H5N1 viruses. The carboxyl terminus of NS1 proteins from most avian influenza virus isolates contain a domain termed the PDZ-binding domain (PDZ-BD) or PDZ-binding motif (PBM) that contributes to virulence by an unknown mechanism. We will study the function and structure of a NS1 protein and its PBM from a H6N6 avian influenza virus.